DESCRIPTION: Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome may underlie the persistence of cocaine-induced addictive-like behaviors. However whether specific changes in chromatin regulation are truly causative for drug-induced behavioral plasticity has remained a challenging hypothesis to test due to the lack of high-throughput in vivo methods for site-specific experimental manipulation of the epigenome. To overcome this limitation we will generate two novel Cre/loxP-conditional CRISPR/Cas9- based transgenic mouse strains in which an enzymatically dead Cas9 protein fused either to the core histone acetyltransferase domain of p300 (dCas-p300) or the KRAB repressor domain (dCas9-KRAB) is knocked into the Rosa26 locus. We have shown that gRNA-mediated recruitment of dCas9 fusions with chromatin regulators is sufficient to induce targeted histone modifications and highly specific corresponding changes in gene transcription. Now by expressing these fusion proteins in transgenic mice, we will achieve regional and temporal control of site-specific epigenome editing in the brain in vivo by intersecting Cre-dependent induction of dCas9-fusion protein expression with stereotaxic viral delivery of validated gRNAs targeting cocaine- regulated enhancers. In the R21 phase of this proposal we will first generate and characterize the conditional dCas9-p300 and dCas9-KRAB mouse strains and then conduct a proof-of-principle experiment to validate whether dCas9-mediated regulation of Fosb in neurons of the nucleus accumbens is sufficient to alter cocaine- induced locomotor sensitization and conditioned place preference. In the R33 phase we will use the dCas9- p300 and dCas9-KRAB mouse strains to test the hypothesis that epigenetic sensitization of Bdnf transcription in dopaminergic neurons of the ventral tegmental area promotes incubation of cocaine craving, an important rodent model for relapse. Finally to accelerate future epigenome editing studies we will first use a novel method to capture nuclei of cocaine-activated neuronal ensembles in the nucleus accumbens for chromatin profiling and then develop and functionally validate gRNAs targeting cocaine-regulated enhancers for use by the broader scientific community. Our studies will provide a novel toolbox for functional epigenomic studies of the molecular mechanisms underlying substance use disorders.